Modified nucleic acid editing systems for tethering donor dna

ABSTRACT

The technology relates to a composition for tethering donor DNA to a nuclease, the composition comprising a nucleic acid comprising donor DNA and a consensus sequence for a DNA binding domain; and at least one of: a fusion protein comprising a nuclease coupled to a DNA binding domain for binding the consensus sequence; and a nucleic acid encoding the fusion protein.

RELATED APPLICATION

This application claims priority to Australian provisional patentapplication No 2018900990 filed 25 Mar. 2018 which is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The technology relates to compositions for tethering donor DNA to anuclease the use of those compositions for improving the efficiency ofin vivo gene editing.

BACKGROUND

A number of genome editing technologies are known includingTranscription Activator-Like Effector Nucleases (TALENs), theCRISPR-Cas9 system (Clustered, Regularly Interspaced, Short PalindromicRepeats and CRISPR associated protein 9), and Zinc finger nucleases(ZFNs). TALENs, CRISPR-Cas9 protein and ZFNs use endonucleases toinitiate double-strand breaks (DSBs) at almost any target sequence ingenomic DNA and can be used for gene knockouts, gene knock-ins, genetagging, and correction of genetic defects.

The type II bacterial clustered, regularly interspaced, shortpalindromic repeats (CRISPR)-associated protein 9 (Cas9) system is atool for the targeted introduction of mutations into cellular DNA.Well-designed single guide (sg) RNAs induce Cas9-mediated doublestranded breaks (DSBs) at desired target sites in cellular DNA whileminimizing effects at other locations. Double stranded breaks stimulateDNA repair by at least two distinct mechanisms non-homologous endjoining (NHEJ) and homology directed repair (HDR). Cas9-mediatedmodification of cellular DNA by NHEJ can reach efficiencies of 20-60%but because NHEJ is error-prone and introduces unpredictable patterns ofinsertions and deletions it is only suitable for introducing smallrandom mutations. Co-application CRISPR-Cas9 with a single-stranded ordouble-stranded DNA template homologous to the sequences flanking thecleavage site on the cellular DNA enables precise genome editing byHDR-mediated incorporation of an exogenous, or donor DNA fragment.However, the frequency of HDR is inherently low and the efficiency ofinsertion of a donor DNA using this strategy is only 0.5-20%.

TALENs are fusions of transcription activator-like (TAL) proteins and aFok I nuclease. TAL proteins are typically composed of 33-34 amino acidrepeating motifs with two variable positions that have a strongrecognition for specific nucleotide sequences. By assembling arrays ofTALs and fusing them to a Fok I nuclease, specific cutting of the genomecan be achieved. When two TALENs bind and meet, the Fok I domains inducea double-strand break which can inactivate a gene, or can be used toinsert DNA of interest. TALENs are able to modify chromosomes withefficiency of up to about 33%.

ZFNs are a class of engineered DNA-binding proteins that allow targetedgenome editing of the genome by creating double-strand breaks in DNA atdesired locations. ZFNs consist of two functional domains, a DNA-bindingdomain comprised of a chain of Zinc-finger modules, each recognizing aunique DNA hexamer. Multiple Zinc-fingers can be assembled to form ZFNwith specificity for a sequence of 24 bases or more. The secondfunctional domain is the nuclease domain of Fok I. Using ZFNs can resultin single or biallelic edits occurring at an efficiency of 1-20% ofclone population.

The present inventor has developed compositions and methods that utilisethe target specificity of gene editing systems such as CRISPR-Cas9,TALENs and ZFNs to tether a donor DNA to a desired target DNA sequence.

SUMMARY

In a first aspect, there is provided a composition comprising acomposition for tethering donor DNA to a nuclease, the compositioncomprising a nucleic acid comprising donor DNA and a consensus sequencefor a DNA binding domain; and at least one of:

a fusion protein comprising a nuclease coupled to a DNA binding domainfor binding the consensus sequence; and

a nucleic acid encoding the fusion protein.

The nuclease may be a Cas, a Transcription activator-like effectornuclease (TALEN), a meganuclease, or a Zinc Finger. In one embodimentthe is a Cas protein, for example Cas9

The fusion protein may further comprises a nuclear localizationsequence.

The composition may further comprise a guide RNA that interacts with theCas protein and a target DNA sequence.

The consensus sequence may comprise the Lac operator (SEQ ID NO: 66),the TRP operator (SEQ ID NO: 68), the TET operator (SEQ ID NO: 67), theGAL-4 binding site (SEQ ID NO: 1), or the IHF binding site (SEQ ID NO2).

The consensus sequence may comprise a sequence with at least 80%, 85%,90%, 95% or at least 99% identity to the Lac operator, the TRP operator,the TET operator, the GAL-4 binding site, or the IHF binding site

The DNA biding domain may comprises the LAC repressor, TET repressor,TRP-repressor, GAL-4, or IHF, or a portion thereof sufficient to bindthe consensus sequence.

The DNA binding domain is the LAC repressor, preferably amino acids43-403 of SEQ ID NO 9.

The nuclease may be coupled to the DNA binding domain via a linker. Thelinker may comprise a sequence selected from any one of SEQ ID Nos: 3 to7, a GGS linker, or amino acids 404-419 of SEQ ID NO 9.

In one embodiment the fusion protein comprises the LAC repressor andCas9.

The composition may comprise a vector, wherein the vector compriseseither or both of:

a. the nucleic acid comprising the donor DNA and the consensus sequencefor a DNA binding domain; and

b. the nucleic acid encoding the fusion protein.

The vector may further comprise a nucleic acid sequence encoding a guideRNA that interacts with the Cas9 and a target DNA sequence.

In some embodiments the fusion protein is nuclease deficient.

In a second aspect there is provided an isolated host cell comprisingthe composition of the first aspect.

In a third aspect there is provided a method for editing DNA in a cell,the method comprising;

contacting the cell with the composition of the first aspect underconditions suitable for the interaction of the fusion protein with atarget DNA sequence.

In a third aspect there is provided a method for editing DNA in a cell,the method comprising

a) contacting the cell with the composition of claim 15 under conditionssuitable for the interaction of the fusion protein with a first targetDNA sequence; and

b) contacting the cell with a nucleic acid editing system adapted toedit the genomic DNA at a second target DNA sequence, under conditionssuitable for nucleic acid editing.

The target DNA sequence may be selected from genomic DNA, mitochondrialDNA, viral DNA, or exogenous DNA.

In one embodiment the efficiency of editing is at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90% at least 95%, or at least 99%.

The cell may be in a subject, preferably a human subject.

In a fourth aspect there is provided a kit comprising:

a nucleic acid comprising donor DNA and a consensus sequence for a DNAbinding domain; and at least one of

-   -   a fusion protein comprising a nuclease coupled to a DNA binding        domain for binding the consensus sequence; and    -   a nucleic acid encoding the fusion protein.

In one embodiment the fusion protein in the kit comprises a Cas protein,a Transcription activator-like effector nuclease (TALEN), ameganuclease, a Zinc Finger or a MADzyme. In one embodiment the Cas isCas9.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this specification.

In order that the present invention may be more clearly understood,preferred embodiments will be described with reference to the followingdrawings and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of two embodiments of the composition disclosedherein comprising a modified nucleic acid editing system.

FIG. 2 is a map of a plasmid vector containing an active Cas 9 genesequence and its guide RNA capable of directing binding of theCrispr/Cas9 complex to a genomic target DNA sequence, donor DNA sequencethat can be used for repair of Crispr/Cas9 induced double strand breaks.

FIG. 3 is a map of a plasmid used in a binary system where theCrispr/Cas9 gene sequence containing the lac repressor fusion and guideRNA are present on a tether expression vector and the donor DNA sequenceis present on a second tethered gene targeting vector.

FIG. 4 is a map of a plasmid used in a binary system where theCrispr/Cas9 gene sequence containing the lac repressor fusion and guideRNA are present on a tether expression vector and the donor DNA sequenceis present on a second tethered gene targeting vector.

FIG. 5 is a map of a plasmid for generating an in vitro system wherebypurified Crispr/Cas9 would is used to bind a gene targeting vector priorto transfection into cells.

FIG. 6 is a map of a plasmid for generating an in vitro system wherebypurified Crispr/Cas9 would is used to bind a gene targeting vector priorto transfection into cells.

FIG. 7 is a workflow diagram for construction of an in vivo tetheredgene-targeting plasmid, where A represents the synthesis of U6expression cassette containing Ug promoter with a central SspIrecognition sequence and flanking SspI compatible overhangs that do notreconstruct the plasmid SspI recognition sequence. B represents thesynthesis of CMV promoter-SV40-polyA cassette containing central NotIcloning site and Eco109I compatible overhangs. C represents Synthesis ofgene encoding Cas9-lac repressor fusion protein with NotI compatibleoverhangs. D represents synthesis of gene encoding RNA complementary togenomic target DNA sequence with SspI compatible overhangs. E representPCR amplification of donor DNA sequence with oligonucleotide containingflanking SaII restriction endonuclease recognition sequences anddigestions with SaII.

FIG. 8A is a workflow diagram for the construction of pGT1. Constructionof lactose repressor gene with inactivated BbsI restriction endonucleasesites.

FIG. 8B is a workflow diagram for the construction of pGT1.

FIG. 8C is a workflow diagram for the construction of pGT1. Threeoverlapping DNA fragments comprising Flag/SV40 NLS, lacI and Cas9 weremade, and these were cloned into pSpCas9 BB-2A-GFP(px458).

FIG. 8D is a vector map if pGT1.

FIG. 9 is a vector map of the ptet repressor plasmid used in theconstruction of pGT9.

FIG. 10A is a workflow diagram for the construction of pGT9. A tetrepressor cassette was cloned into pUC57 to create ptet repressor.

FIG. 10B is a workflow diagram for the construction of pGT9. Threepolymerase chain reaction products, Flag/SV40 NLS, lacI and Cas9, weregenerated.

FIG. 10C is a workflow diagram for the construction of pGT9. Threepolymerase chain reaction products, Flag/SV40 NLS, lacI and Cas9 werecloned into pSpCas9 BB-2A-GFP(px458).

FIG. 10D is a vector map of pGT9.

FIG. 11 is an overview of a donor fragment, showing a 500 pase pairdonor fragment with deIF508 Mutation and lacO or tetO RecognitionSequences

FIG. 12 is an overview of the use of the compositions described hereinfor homology directed repair by homologous recombination between thegenomic CFTR target Cas9-induced double strand break and donor fragmentto catalyze transfer of the deIF508 DNA sequence to the genomic target.

FIG. 13 illustrates that combination of pGT1 vector (lactoserepressor-Cas9 fusion) with the 130117 guide (genomic target forwardstrand) and lactose operator on the 3′ end of donor DNA (FIG. 13, lane7) demonstrates higher gene editing efficiency as compared to the px458vector with unmodified Cas9 and same donor DNA fragment

FIG. 14 illustrates that gene editing with the pGT9 tetracyclinerepressor-Cas9 fusion vector, guide sequences, and donor fragments didnot yield appreciably better gene editing frequencies than the controlpx458 vector

DESCRIPTION OF EMBODIMENTS

The present disclosure provides compositions, methods, and kits fortethering donor DNA to a DNA target sequence with a modified nucleicacid editing system. The disclosure provides for improved efficiency ofin vivo cellular DNA modification (such as gene editing) using amodified nucleic acid editing system, such as CRISPR-Cas9. Oneembodiment of this process is illustrated in FIG. 1. The modified geneediting system is adapted a bind to a nucleic acid comprising donor DNAand tether the nucleic acid to a specific site on the cellular DNA at ornear to the nucleotide sequence to be edited.

Modified Nucleic Acid Editing Systems (Nuclease-DNA Binding DomainFusions)

The compositions and methods described herein can include a nuclease ofany nucleic acid editing system capable of site specific binding. Forexample, useful nucleases include Cas nucleases, TALENs, meganucleases,ZFNs and MADzymes. The nuclease is modified by combining it with a DNAbinding domain.

The nuclease and the DNA binding domain may be joined via linker.Suitable linkers include for example, linkers comprising the sequences(Gly-Gly-Gly-Gly-Ser)_(n), (Gly)_(n), or (Gly)_(n)S, (EAAAK)_(n),(AP)_(n), (XP)_(n), or A(EAAAK)_(n) where n is any one of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and X is anyamino acid. Other suitable linkers comprise the sequencesKESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, KESGSVSSEQLAQFRSLE, orGGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG.

Cas systems are divided into three major types (type I, type II, andtype III) and twelve subtypes, which are based on their genetic contentand structural differences. However, the core defining features of allCRISPR-Cas systems are the Cas genes and their proteins: cas1 and cas2are universal across types and subtypes, while Cas3, Cas9, and Cas10 aresignature genes for type I, type II, and type III, respectively.

Any Cas may be used. For example the Cas nuclease may be Cas1, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10. In some embodiments theCas is Cas9.

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA nuclease used toinduce site-directed double strand breaks in DNA for the geneinactivation or the introduction of heterologous genes throughnon-homologous end joining and homologous recombination respectively.Systems and nucleic acids sequences for expressing Cas9 are commerciallyavailable. The Cas9 may be codon optimized for a human or othermammalian system. The Cas9 protein may contain a nuclear localizationsignal at the C-terminus. The RNA encoding Cas9 may be capped andpolyadenylated to support expression in mammalian cells, and may containmodifications to reduce immune stimulation. The amino acid sequence andencoding nucleic acid sequence for Cas9 and functional derivatives andhomologs which can be used in the compositions and methods are known inthe art.

The Cas9 may be delivered in conjunction with a guide RNA (gRNA) thatdirects the editing system to the nucleotide sequence recognized by thegRNA. In general, a gRNA can be designed to target any nucleotidesequence. The gRNA structure is disclosed in, for example, Ran F A,Genome editing using the CRISPR-Cas9 System. PNAS 8(1 1):2281-308(2013); and Pyzocha et al., RNA-guided genome editing of mammaliancells. Methods Mol. Biol. 1 1 14:269-77 (2014), which are herebyincorporated by reference in their entirety. Generally for Cas9, gRNAsguide the Cas9 to the complementary 20 nucleotide sequences with adownstream NGG protospacer-adjacent motif (PAM).

The CRISPR-Cas9 system including the construction of guide sequences isfurther disclosed in U.S. Pat. No. 8,697,359, which is herebyincorporated by reference in its entirety.

In place of a CRISPR-Cas9 system, alternate nucleic acid editing systemsmay be used. For example, suitable systems include any CRISPR/cas system(e.g., any Cascade-like CRISPR/cas, Type I CRISPR cas, Type IICRISPR/cas, and type III CRISPR/cas), zinc finger nucleases (ZFNs),transcription activator-like effector nucleases (TALENs), meganucleases,homing endonucleases, and MADzymes (for examples the Human or E. coliMADzymes encoded by SEQ ID Nos: 69 and 70, respectively.

The DNA binding domain component of the modified nucleic acid editingsystem can be any sequence specific high affinity DNA binding domain.The term ‘DNA binding domain’ as used herein refers to any completeprotein or fragment thereof which can bind DNA. Accordingly the term‘DNA binding domain’ includes complete proteins such as the LACrepressor and fragments of the protein which retain the DNA binding

In some embodiments the DNA binding domain is not mammalian in order toreduce of target effects. For example the DNA binding domain may be froma bacteria or yeast. DNA binding domains useful in the compositions andmethods described herein can be selected from the group consisting of bethe LAC repressor, TET repressor, TRP-repressor, GAL-4, or IHF. Each ofthese bind specific consensus sequences that are known in the art.

The consensus sequence may be the Lac operator (e.g. SEQ ID NO: 66), theTRP operator (e.g. SEQ ID NO: 68), the TET operator (e.g. SEQ ID NO:67), the GAL-4 binding site (5′-CGG-N₁₁-CCG-3′) or the IHF binding site(5′-WATCAANNNNTTR-3′). W is A or T, and R is A or G, and N is anynucleotide.

The nucleic acid editing systems can be present in the compositionsdisclosed herein in the form of purified proteins or in the form ofnucleic acids encoding the nucleic acid editing system. The nucleic acidmay be a vector, for example a plasmid vector comprising sequencesencoding the nucleic acid editing system operably linked to aconstitutive or inducible promoter.

The term “vector” includes a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsused to deliver the nucleic acids to cells as described herein includevectors known to those of skill in the art and used for such purposes.Certain exemplary vectors may be plasmids, lentiviruses oradeno-associated viruses. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g. circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.retroviruses, lentiviruses, replication defective retroviruses,adenoviruses, replication defective adenoviruses, and adeno-associatedviruses). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g. bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are expression vectors capable ofdirecting the expression of genes to which they are operatively linked.Common expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids. Recombinant expression vectors cancomprise one or more nucleic acids encoding a modified nucleic acidediting system in a form suitable for expression of the nucleic acid ina cell, which means that the recombinant expression vectors include oneor more regulatory elements, which may be selected on the basis of thehost cells to be used for expression. That is the regulatory elementsare operatively-linked to the nucleic acid sequence to be expressed.‘Operably linked’ means that that the nucleotide sequence of interest islinked to the regulatory element(s) in a manner that allows forexpression of the nucleotide sequence (e.g. in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell).

‘Regulatory element’ includes promoters, enhancers, internal ribosomalentry sites (IRES), and other expression control elements (e.g.transcription termination signals, such as polyadenylation signals andpoly-U sequences). Regulatory elements are known in the art. Regulatoryelements include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes).

Regulatory elements may also direct expression in a temporal-dependentmanner, such as in a cell-cycle dependent or developmentalstage-dependent manner, which may or may not also be tissue or cell-typespecific. In some embodiments, a vector may comprise one or more poI Illpromoter (e.g. 1, 2, 3, 4, 5, or more poI III promoters), one or morepoI H promoters (e.g. 1, 2, 3, 4, 5, or more poI H promoters), one ormore poI I promoters (e.g. 1, 2, 3, 4, 5, or more poI 1 promoters), orcombinations thereof. Examples of poI III promoters include, but are notlimited to, U6 and HI promoters. Examples of poI II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer), the SV40 promoter, thedihydrofolate reductase promoter, the β-actin promoter, thephosphoglycerol Kinase (PGK) promoter, and the EFIa promoter and Poi Hpromoters described herein. Also encompassed by the term “regulatoryelement” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′segment in LTR of HTLV-1; SV40 enhancer; and the intron sequence betweenexons 2 and 3 of rabbit β-globin. It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression desired, etc. A vector can be introduced into hostcells to thereby produce transcripts (e.g. guide RNA), proteins, orpeptides, including fusion proteins (such as the modified nucleic acidediting systems) or peptides.

The vector may include one or more terminator sequences. A terminatorsequence includes a section of nucleic acid sequence that marks the endof a coding sequence during transcription.

The vector may include one or more sequences encoding an epitope tag orreporter gene sequences, Non-limiting examples of epitope tags includehistidine (His) tags, V5 tags, FLAG tags, influenza hemaglutinin (HA)tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples ofreporter genes include, but are not limited to,glutathione-S-transferase (GST), horseradish peroxidase (HAP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,betaglucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and auto-fluorescent proteins including blue fluorescent protein (BFP).The epitope tag, reporter gene or both may be expressed from the vectoras a fusion with the modified nucleic acid editing system (nuclease-DNAbinding domain fusion).

Alternatively or in addition the compositions may comprise a mRNAencoding the fusion protein.

The mRNA can be modified, and the modification selected from one or moreof modifications of the phosphate backbone (e.g., phosphorothioatelinkages or boranophosphate linkages), ribose ring modifications such as2′-0-methyl and/or 2′-fluoro and/or 4′-thio modifications, and locked orunlocked nucleic acids. Other modifications may include pseudouridine,2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine,5-aminouridine, 5-methyluridine, 2-thiopseudouridine,4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine,5-aminopseudouridine, pseudoisocytidine, 5-methylcytidine,N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine,5-aminocytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine,5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine,5-methylpseudoisocytidine, N6-methyladenosine, 7-deazaadenosine,6-thioguanosine, 7-deazaguanosine, 8-azaguanosine,6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine,and 6-thio-7-deaza-8-azaguanosine.

In some embodiments the modifications are selected for one or more ofthe following: reduce immune stimulation, RNA stabilization, improveexpression of the encoded protein. For example, the RNA may have acombination of 2-thiouridine and 5-methyl-cytidine to reduce immunestimulation through pattern recognition receptors such as TLR3, TLR7 andTLR8. In some embodiments, the mRNA has one or more pseudouridine tostabilize the mRNA against cleavage, and improve expression rates.

The modified nucleic acid editing systems also comprise a DNA bindingdomain. The DNA binding domain facilitates the localisation of themodified nucleic acid editing system to cellular DNA.

Construction of the vectors disclosed herein is by standard methodsknown in the art such as ligation of synthetic nucleic acids, or nucleicacids produced by, for example PCR, into a plasmid that has been cut byone or more site-specific nucleases.

There are alternative strategies for producing the vectors disclosedherein. In one approach, the entire vector(s) are synthesized de novousing a commercially available service, for example by a company thatspecialises in the synthesis of large DNA molecules.

Alternatively, a combination of classical cloning techniques andsynthetic biology to modify a standard laboratory plasmid such as pUC19.In this approach, gene cassettes encoding the Cas9-lac operon fusiongene, guide RNA, and donor DNA sequences would be synthesized de novoand individually cloned into unique restriction endonuclease sitespresent in the vector backbone using T4 DNA ligase. Donor DNA sequencesare amplified by polymerase chain reaction to generate products that arecloned into the vector backbone. Modification of Cas9-lac repressorfusion proteins to add other DNA binding domains or epitopes foraffinity purification of modified Cas9 proteins can be performed byrecombinant PCR followed by ligation into restriction endonuclease sitesin the vector backbone. A workflow illustrating this process forpreparing the plasmid vectors disclosed herein is shown in FIG. 7.

Nuclease Deficiency

in some embodiments the nucleic acid editing system is nucleasedeficient. In these embodiments the nuclease deficient nucleic acidediting system is used to tether the donor DNA near to a target nucleicacid sequence so that donor DNA is available for use by an additionalnucleic acid editing system.

For example the nuclease deficient gene editing system has reduced oreliminated nuclease activity, alternatively the nuclease activity isabsent or substantially absent within levels of detection. In someembodiments the nuclease activity of the gene editing system may beundetectable using known assays, i.e. below the level of detection ofknown assays.

Nuclease deficient gene editing systems can be prepared by those skilledin the art using standard molecular biology techniques. Typically thisinvolves deleting or altering one or more amino acids crucial fornuclease activity to substantially eliminate or eliminate nucleaseactivity.

In some embodiments the Cas9 is a nuclease-deficient Cas9. Anuclease-deficient Cas9 may be one in which one or more amino acids inCas9 are altered or removed. For example a nuclease deficient Cas9 maybe generated by removing or mutating one or more of the amino acids D10,H840, D839 and N863 (See Jinke et al., Science 337, 816-821 (2012). Forexample one or more of these amino acids may be deleted or substitutedwith alanine or glycine to substantially eliminates or eliminatesnuclease activity.

Donor DNA

The term “donor DNA” includes a nucleic acid sequence which is to beinserted into cellular DNA (such as of genomic DNA, mitochondrial DNA,or viral DNA). The donor nucleic acid sequence may be expressed by thecell. The donor nucleic can be exogenous, foreign to the cell ornon-naturally occurring within the cell.

The donor DNA is associated with a sequence that can be bound by a DNAbinding domain. For example the donor DNA may be contiguous with aconsensus sequence for a DNA binding domain or may be present on thesame vector as the a consensus sequence, or may be present on the samepolynucleotide as the consensus sequence.

Target Nucleic Acid Sequence

A target nucleic acid sequence includes any nucleic acid sequence, suchas a genomic nucleic acid sequence or a gene to which a nuclease of anucleic acid editing system can co-localise. Target nucleic acidsinclude nucleic acid sequences capable of being expressed into proteins.According to one aspect, the target nucleic acid is genomic DNA,mitochondrial DNA, plastid DNA, viral DNA, or exogenous DNA.

One of skill in the art will readily be able to identify or design guideRNAs, TALENs, ZFNs, meganucleases and homing endonucleases whichco-localize to a target nucleic acid sequence.

Methods

The compositions and vectors described herein can be used in for editingDNA in a cell. The methods comprise contacting the cell with acomposition comprising a modified gene editing system or vector encodinga modified gene editing system under conditions suitable for theinteraction of the modified nucleic acid editing system with a targetDNA sequence. The methods also comprise use of a nuclease-deficientmodified nucleic acid editing system that interacts with a first targetDNA sequence and a conventional nucleic acid editing system to edit theDNA at a second target DNA sequence.

In order to increase the efficiency of DNA editing the donor DNA ispositioned close to the target DNA sequence so that it is readilyavailable for nucleic acid editing.

In embodiments using a nuclease-deficient modified nucleic acid editingsystem this is achieved by spacing the first and second target sequencesso that the donor DNA is closed to the conventional nucleic acid editingsystem when it is colocalised with its target sequence.

For example the first and second target sequences are about 75 to 150base pairs apart, about 150-250, 250-350, 450-550, 550-650, 650-750,750-850, 850-950, 950-1050 base pairs apart or about 1-1.5 kb apart.

The methods require delivery of the compositions or vectors to the cell.Methods of non-viral delivery of nucleic acid vectors, RNA or proteinsinclude lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, agent-enhanced uptake of DNA, nanoparticles, andelectroporation/nucleofection. Lipofection reagents are soldcommercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutrallipids that are suitable for efficient receptor-recognition lipofectionof polynucleotides are known. Delivery can be to cells (e.g. in vitro orex vivo administration) or target tissues (e.g. in vivo administration).The term native includes the protein, enzyme, RNA, or guide RNA speciesitself as well as the corresponding nucleic acid encoding the same.

Delivery Vehicles

Delivery vehicles for the compositions and vectors disclosed hereinprovided herein may be viral vectors or non-viral vectors. In someembodiments, the modified nucleic acid editing system is provided in aviral vector or a non-viral vector. In other embodiments, the guide RNAis provided in a viral vector, and the modified nucleic acid editingsystem is provided in a non-viral vector. In still other embodiments,the guide RNA is provided in a non-viral vector and the modified nucleicacid editing system is provided in a viral vector

In some embodiments, the viral vector is selected from anadeno-associated virus (AAV), adenovirus, retrovirus, and lentivirusvector. While the viral vector may deliver any component of the systemdescribed herein so long as it provides the desired profile for tissuepresence or expression, in some embodiments the viral vector providesfor expression of one or more of the modified nucleic acid editingsystem, guide RNA and optionally the delivers the donor DNA. In someembodiments, the viral delivery system is adeno-associated virus (AAV)2/8. However, in various embodiments other AAV serotypes are used, suchas AAV1, AAV2, AAV4, AAV5, AAV6, and AAV8. In some embodiments, AAV6 isused when targeting airway epithelial cells, AAV7 is used when targetingskeletal muscle cells (similarly for AAV1 and AAV5), and AAV8 is usedfor hepatocytes. In some embodiments, AAV1 and 5 can be used fordelivery to vascular endothelial cells. Further, most AAV serotypes showneuronal tropism, while AAV5 also transduces astrocytes. In someembodiments, hybrid AAV vectors are employed. In some embodiments, eachserotype is administered only once to avoid immunogenicity. Thus,subsequent administrations employ different AAV serotypes.

In some embodiments, the delivery system comprises a non-viral deliveryvehicle. In some aspects, the non-viral delivery vehicle is lipid-based.In other aspects, the non-viral delivery vehicle is a polymer. In someembodiments, the non-viral delivery vehicle is biodegradable. Inembodiments, the non-viral delivery vehicle is a lipid encapsulationsystem and/or polymeric particle.

In certain embodiments, the delivery system comprises lipid particles.In some embodiments, the lipid-based vector is a lipid nanoparticle,which is a lipid particle between about 1 and about 100 nanometers insize. In some embodiments, the lipid-based vector is a lipid orliposome. Liposomes are artificial spherical vesicles comprising a lipidbilayer.

The lipid-based vector can be a small nucleic acid-lipid particle(SNALP). SNALPs comprise small (less than 200 nm in diameter)lipid-based nanoparticles that encapsulate a nucleic acid. In someembodiments, the SNALP is useful for delivery of an RNA molecule. Insome embodiments, SNALP formulations deliver nucleic acids to aparticular tissue in a subject, such as the liver.

In some embodiments, the guide RNA, the modified nucleic acid editingsystem (or the RNA encoding the same) is delivered via polymericvectors. In some embodiments, the polymeric vector is a polymer orpolymerosome. Polymers encompass any long repeating chain of monomersand include, for example, linear polymers, branched polymers,dendrimers, and polysaccharides. Linear polymers comprise a single lineof monomers, whereas branched polymers include side chains of monomers.Dendrimers are also branched molecules, which are arranged symmetricallyaround the core of the molecule. Polysaccharides are polymericcarbohydrate molecules, and are made up of long monosaccharide unitslinked together. Polymersomes are artificial vesicles made up ofsynthetic amphiphilic copolymers that form a vesicle membrane, and mayhave a hollow or aqueous core within the vesicle membrane.

Various polymer-based systems can be used for administering RNA encodingmodified nucleic acid editing system. Exemplary polymeric materialsinclude poly(D,L-lactic acid-co-glycolic acid) (PLGA),poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA),poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid)(PGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide) (PLLA), PLGA-b-poly(ethylene glycol)-PLGA(PLGA-bPEG-PLGA), PLLA-bPEG-PLLA, PLGA-PEG-maleimide (PLGA-PEG-mal),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)(polyacrylic acids), and copolymers and mixtures thereof, polydioxanoneand its copolymers, polyhydroxyalkanoates, polypropylene fumarate),polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid),poly(valeric acid), poly(lactide-co-caprolactone), trimethylenecarbonate, polyvinylpyrrolidone, polyortho esters, polyphosphazenes,Poly([beta]-amino esters (PBAE), and polyphosphoesters, and blendsand/or block copolymers of two or more such polymers. Polymer-basedsystems may also include Cyclodextrin polymer (CDP)-based nanoparticlessuch as, for example, CDP-admantane (AD)-PEG conjugates andCDP-AD-PEG-transferrin conjugates.

In one embodiment, nanoparticles are formulated with Cas9 mRNAchemically modified to reduce TLR responses, as disclosed in Kormann etal. Expression of therapeutic proteins after delivery of chemicallymodified mRNA in mice. Nat. Biotechnol. 29: 154-157 (2011). In a furtherembodiment, the nanoparticles are formulated using controlledmicrofluidic mixing systems, as disclosed in, for example, Chen et al.Rapid discovery of potent siRNA-containing lipid nanoparticles enabledby controlled microfluidic formulation. J. Amer. Chem. Soc.134:6948-6951 (2012).

In some embodiments, the lipid-based delivery system comprises a lipidencapsulation system. The lipid encapsulation system can be designed todrive the desired tissue distribution and cellular entry properties, aswell as to provide the requisite circulation time and biodegradingcharacter. The lipid encapsulation may involve reverse micelles and/orfurther comprise polymeric matrices. In some embodiments, the particleincludes a lipophilic delivery compound to enhance delivery of theparticle to tissues, including in a preferential manner. Such compoundsmay generally include lipophilic groups and conjugated amino acids orpeptides, including linear or cyclic peptides, and including isomersthereof.

The lipid or polymeric particles may have a size (e.g., an average size)in the range of about 50 nm to about 5 μm. In some embodiments, theparticles are in the range of about 10 nm to about 100 μm, or about 20nm to about 50 μm, or about 50 nm to about 5 μm, or about 70 nm to about500 nm, or about 70 nm to about 200 nm, or about 50 nm to about 100 nm.Particles may be selected so as to avoid rapid clearance by the immunesystem. Particles may be spherical, or non-spherical.

In some embodiments, the non-viral delivery vehicle may be a peptide,such as a cell-penetrating peptides or cellular internalizationsequences. Cell penetrating peptides are small peptides that are capableof translocating across plasma membranes. Exemplary cell-penetratingpeptides include, but are not limited to, Antennapedia sequences, TAT,HIV-Tat, Penetratin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP(model amphipathic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB I,Pep-7, I-IN-1, BGSC (Bis-Guanidinium-Spermidine-Cholesterol, and BGTC (Bis-Guanidinium-Tren-Cholesterol).

In some embodiments, the guide RNA or RNA encoding the modified nucleicacid editing system is modified at the 5′ end or the 3′ end In apreferred embodiment, the modification is made at the 3′ end of the RNA.The RNA may be modified by conjugating to cholesterol, other lipophilicmolecules, polymers, peptides, antibodies, aptamers, and/or smallmolecules. In some embodiments, the RNA is conjugated to aN-acetylgalactosamine (GaINAc). GaINAc binds the asialoglycoproteinreceptor (ASGPR) on hepatocytes, and therefore can be used to target anRNA to the liver. In some embodiments, the RNA is conjugated to atrivalent targeting ligand, e.g., triantennary GaINAc. Such conjugatescomprise an RNA conjugated at the 3′ terminus to three GaINAc molecules.

The delivery vehicles (e.g. conjugates, viral or non-viral vectors, orany combination thereof) may be administered by any method known in theart, including injection, optionally by direct injection to targettissues. In some embodiments, the guide RNA, modified nucleic acidediting system, and, optionally, donor DNA are administeredsimultaneously in the same or in different delivery vehicles. In otherembodiments, the guide RNA and modified nucleic acid editing system and,optionally, donor DNA are administered sequentially via the same orseparate delivery vehicles. In some embodiments, the guide RNA and/ordonor DNA is administered 1, 3, 5, 7, 10, 14, or 30 days prior toadministration of the modified nucleic acid editing system, such thatthe guide RNA and/or donor DNA accumulates in the target cell or tissueprior to administration of the modified nucleic acid editing system. Insome embodiments, the guide RNA, donor DNA and/or nucleic acid editingsystem is administered in a plurality of doses, such as 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses. Invarious embodiments, the gRNA, donor DNA and/or nucleic acid editingsystem is administered over a time period of from one day week to abouta month.

In one embodiment, one or both of the guide RNA and donor DNA, areprovided in an AAV vector that is administered to the tissue or cellprior to administration of the modified nucleic acid editing system. Ina further embodiment, the AAV vector comprising the gRNA is administered1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to the administration of thenanoparticle modified nucleic acid editing system, to allow expressionof the guide RNA from the AAV vector. In a yet further embodiment, themodified nucleic acid editing system is administered multiple times, forexample, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15days.

In another embodiment, the donor DNA is delivered via an AAV vector, andis injected 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to theadministration of either or both of the modified nucleic acid editingsystem and the guide RNA.

In particular embodiments, either or both of guide RNA and donor DNA areprovided in an AAV vector that is administered first, and the modifiednucleic acid editing system is administered subsequently in alipid-based delivery vehicle in one or more doses.

In another embodiment, each component of the compositions describedherein (e.g., the modified nucleic acid editing system, guide RNA anddonor DNA) are each delivery using a different vehicles, alternativelyone or more components may be used with the same deliver vehicle. In afurther embodiment, the modified nucleic acid editing system, guide RNA,and donor DNA, are administered at multiple time points, for example,every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days. Inanother embodiment, the administration of the modified nucleic acidediting system, guide RNA and donor DNA are administered at differenttime points.

In some embodiments, expression of the modified nucleic acid editingsystem is transient. In some embodiments, such transient expression ofthe modified nucleic acid editing system minimizes off-target effects.For example, expression of the modified nucleic acid editing system iscontrolled via selection of the delivery vehicles and/or promoters.

In some embodiments, the present disclosure provides compositions andmethods that allow for increased safety and/or efficacy of conventionalnucleic acid editing systems. Advantageously, the methods disclosedherein provide for repeated dosing with conventional and modifiednucleic acid editing systems such that the efficiency of gene editingincreases with each dose. For example, in some embodiments, the methodsdisclosed herein result in an increase in efficiency of gene editing byconventional nucleic acid editing systems when used in conjunction withthe modified nucleic acid editing systems disclosed herein. For examplethe percentage efficiency of gene editing increases by about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%,about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%,about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%,about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%,about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%,about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about74%, about 75%, or more.

In another embodiment at least one of the guide RNA, modified nucleicacid editing system, and donor DNA are administered to a tissue or cellat the same time, such as on the same delivery vehicle, and one or morecomponent (i.e., the modified nucleic acid editing system, and guideRNA) is under the control of an inducible promoter. As an example, inone embodiment the inducible promoter may be for example a smallmolecule-induced promoter such as tetracycline-inducible promoter.

The delivery vehicles (whether viral vector or non-viral vector or RNAconjugate material) may be administered by any method known in the art,including injection, optionally by direct injection to target tissues orcells. Nucleic acid modification can be monitored over time by, forexample, periodic biopsy with PCR amplification and/or sequencing of thetarget region from genomic DNA, or by RT-PCR and/or sequencing of theexpressed transcripts. Alternatively, nucleic acid modification can bemonitored by detection of a reporter gene or reporter sequence.Alternatively, nucleic acid modification can be monitored by expressionor activity of a modified gene product or a therapeutic effect in thecell or tissue or in a subject.

In some embodiments, the cell or tissue is in a subject. For example thesubject may be a human, in particular a human in need of therapeutic orprophylactic intervention. Alternatively, the subject is an animal,including livestock, poultry, domesticated animal, or laboratory animal.In various embodiments, the subject is a mammal, such as a human, horse,cow, dog, cat, rodent, or pig.

In some embodiments, the methods provided herein include obtaining acell or population of cells from a subject and modifying a targetpolynucleotide in the cell or cells ex vivo, using the delivery systems,compositions, methods, and/or kits disclosed herein. In furtherembodiments, the ex vivo modified cell or cells may be re-introducedinto the subject following ex vivo modification. Thus, the presentdisclosure provides methods for treating a disease or disorder in asubject, comprising obtaining one or more cells from the subject,modifying one or more target nucleotide sequences in the cell ex vivousing both conventional and the modified nucleic acid editing systemsdescribed herein and re-introducing of the cell with the modified targetnucleotide sequence back into the subject having the disease ordisorder. In some embodiments, cells in which nucleotide sequencemodification has occurred are expanded in vitro prior to reintroductioninto the subject having the disease or disorder.

In other embodiments, at least one of the modified nucleic acid editingsystem, guide RNA and donor DNA are administered to a cell in vitro.

In some embodiments, at least one component (e.g., the guide RNA, donor,modified nucleic acid editing system or nucleic acid vector) accumulatesin a cell or tissue which may be, for example, liver, heart, lung(including airway epithelial cells), skeletal muscle, CNS (e.g., nervecells), endothelial cells, blood cells, bone marrow cells, blood cellprecursor cells, stem cells, fat cells, or immune cells. Tissuetargeting or distribution can be controlled by selection and design aviral delivery vehicle, or in some embodiments is achieved by selectionand design of lipid or polymeric delivery vehicles.

In some embodiments, the percentage efficiency of target sequencemodification (editing) using the methods disclosed herein is at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% at least 95%, or at least99%.

In some embodiments, the efficiency of target sequence modification(editing) using the methods and compositions disclosed herein provides a2-fold, 3-fold, 4-fold, 5-fold, 6-fold, a 7-fold increase or morecompared to methods of making the same modification (edit) withouttethering the donor DNA.

In some embodiments, the efficiency of target sequence modification isless than 100%, or wherein an effect on fewer than 100% of the cells hasa therapeutic effect. For example, a therapeutic effect may be achievedwhen the efficiency of nucleic acid modification of about 0.01% to about100%, about 0.01% to about 50%, about 0.05% to about 40%, about 0.1% toabout 30%, about 0.5% to about 25%, about 1% to about 20%, about 1% toabout 15%, about 1% to about 10%, or about 1% to about 5%. Thus, even ifthe efficiency of nucleotide sequence modification is relatively low(e.g., less than 50%, or less than 40%, or less than 30%, or less than20%, or less than 10%, or less than 5%, or less than 1%, or less than0.5%, or less than 0.1%), modest expression of the introduced orcorrected or modified gene product may result in a therapeutic effect inthe disease or disorder.

In some embodiments, the delivery systems and compositions disclosedherein are formulated such that the ratio of the components is optimizedfor consistent delivery to the target sequence. In one embodiment, theratio of the guide RNA and modified nucleic acid editing system isoptimized for consistent delivery to the target sequence. In anotherembodiment, the ratio of the donor DNA to the guide RNA and/or to themodified nucleic acid editing system is optimized for consistentdelivery to the target sequence. For example, in one embodiment, theratio of modified Cas9:guideRNA:donor is from about 1:1:1 to about1:1:100. In a further embodiment, the ratio is from about 1:1:2 to about1:1:90, from about 1:1:5 to about 1:1:75, or from about 1:1:10 to about1:1:50. In other embodiments, the ratio is about 1:1:1 or below, such asfrom about 1:1:0.01 to about 1:1:1, from about 1:1:0.02 to about1:1:0.75, or about 1:1:0.05 to about 1:1:0.5, or about 1:1:0.1 to about1:1:0.5. In other embodiments, wherein the composition does not includea guide RNA, the ratio of modified nucleic acid editing system:donor DNAis from about 1:100 to about 100:1, or about 1:50 to about 50:1, orabout 1:25 to about 25:1, or about 1:10 to about 10:1, or about 1:5 toabout 5:1, or about 1:2 to about 2:1, or about 1:1.

Kits

In one aspect, there is provides kits containing any one or more of thecomponents disclosed in the above methods, compositions, and deliverysystems. Kit components may be provided individually or in combinations,and may be provided in any suitable container, such as a vial, a bottle,or a tube. In some embodiments, the kits disclosed herein comprise oneor more reagents for use in the embodiments disclosed herein. Forexample, a kit may provide one or more reaction or storage buffers.Reagents may be provided in a form that is usable in a particularmethod, or in a form that requires addition of one or more othercomponents before use (e.g. in concentrate or lyophilized form).Suitable buffers include, but are not limited to, phosphate bufferedsaline, sodium carbonate buffer, sodium bicarbonate buffer, boratebuffer, Tris buffer, MOPS buffer, HEPES buffer, and combinationsthereof. In some embodiments, the buffer is alkaline. In someembodiments, the buffer has a pH from about 7 to about 10.

For example, a kit may comprise: a donor DNA and a modified nucleic acidediting system. The kit may further comprise a guide RNA. The kit mayprovide an expression system providing for expression of either or bothof the modified nucleic acid editing system and guide RNA in a targetcell. The kit may provide one or more doses of an RNA delivery system,each dose providing for expression of the modified nucleic acid editingsystem in the target cell or tissue.

The kit may be custom made for use with user defined target sequences.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

EXAMPLES Example 1: Plasmid for Tethering Crispr/Cas9 Complex to aGenomic Target DNA Sequence

FIG. 2 is a map of a plasmid vector containing an active Cas 9 genesequence and its guide RNA. The coding sequence of Cas9 is contiguouswith a lac repressor DNA binding domain. This fusion is operably linkedto a CMV promoter. When expressed lac repressor DNA binding domain bindsthe lac operator sequence in the plasmid backbone sequence.

Donor DNA complementary to a genomic target DNA sequence is also clonedinto the vector and provides a template for homologous recombinationbetween the Crispr/Cas9 generated double-strand break in the target DNAsequence. In one embodiment the donor DNA sequence is modified toprevent binding of the Crispr/Cas9 nuclease to plasmid sequence andcontains selectable markers to aid in identification of recombinant celllines. It is contemplated that the donor DNA sequence may contain mutantDNA sequences to change the function of the target chromosomal gene orit may contain a ‘wild type’ sequence to correct a mutant target DNAsequence.

Example 2: Plasmids for a Binary Tethering System

FIGS. 3 and 4 are maps of plasmids used in a binary system where theCrispr/Cas9 gene sequence containing the lac repressor fusion and guideRNA are present on a tether expression vector (FIG. 3) and the donor DNAsequence is present on a second tethered gene targeting vector (FIG. 4).Co-transfection of both vectors is necessary for expression of thetarget-specific Crispr/Cas9/lac repressor nuclease binds the lacoperator sequence on the tethered targeting plasmid (FIG. 4) thuslocalizing the gene targeting plasmid to the target DNA.

The sequences of complementary oligonucleotides used to clone thelactose operator sequence or tetracycline resistance operator sequenceinto HindIII/SaII restriction endonuclease digested pUC19. The correctorientation allows subsequent cloning of LacO and TetO duplexes intopUC19 to generate plasmids with one or more sequential operators thatcan be used to clone donor DNA molecules for gene editing by homologydirected recombination in mammalian cells.

TABLE 1 LacO and TetO Oligonucleotides. SEQ Name Sequence ID NO tetO wTCGAGTTTACCACTCCCTATCAGTGATAGAG 59 AAAAGTGAAAG tetO cTCGACTTTCACTTTTCTCTATCACTGATAGG 60 GAGTGGTAAAC lacO wTCGAGTTTAGTGGAATTGTGAGCGGATAACA 61 ATTTCACTGAAAG lacO cTCGACTTTCAGTGAAATTGTTATCCGCTCAC 62 AATTCCACTAAAC

Example 3: Plasmids for an In Vitro Tethering System

FIGS. 5 and 6 are plasmid maps of vectors for an in vitro tetheringsystem whereby purified Crispr/Cas9 is used to bind a gene targetingvector prior to transfection into cells. The His-tagged Crispr/Cas9-lacrepressor fusion is expressed from the plasmid shown in FIG. 5, thefusion is then purified by immobilized metal affinity chromatography.FIG. 5 indicates that the Lac represser protein operator-binding domainis fused to the c-terminus of the CAS9 9. However, as set out in SEQ IDNOS: 8 and 9 a preferable arrangement is that the lac repressor is fusedto the N-terminus of Cas9 preferably using a linker such as the XTENlinker. In any case the purified fusion protein is the mixed with a genetargeting vector (i.e. FIG. 4). The complex of purified fusion proteinand gene targeting vector is transfected into cells with the guide RNAexpression plasmid (FIG. 6).

Example 4. Gene Editing of the Cystic Fibrosis Transmembrane ConductanceRegulator (CFTR) Gene by GeneTethered Donor DNA Fragments

Cystic fibrosis is one of the most prevalent genetic diseases found inthe Caucasian population with as many as 1/27 individuals carrying amutation in the CFTR gene. The most common mutation found in CF patientsis the deIF508, or F508del, mutation that is the result of an in frame 3base pair deletion in exon 11 resulting in a loss of phenylalanine atresidue 508 in the protein. Several immortalized human cell lines havebeen generated from CF patients, including the CFBE41o-cell linehomozygous for the deIF508 mutation. The CFTR gene sequence in HEK293cells, however, is a normal, or wild type, CFTR gene sequence.

In this example, the px458 Cas9 vector was modified by engineering alactose repressor protein, or tetracycline repressor protein, fused inframe with the Cas9 protein sequence to create a “GeneTether”. ThisGeneTether modification enables binding of donor DNA molecules used forgene editing to the functional Cas9 protein and localizes the donor DNAmolecule at the Cas9-generated double strand break to enhance homologydirected repair, reduce on-target mutations, and reduce induction of theP53 DNA repair system. These modified Cas9 proteins were transfectedinto cells with donor DNA molecules containing lactose operator ortetracycline operator DNA sequences to measure the gene editingefficiency introducing the deIF508 mutation into the normal HEK293 CFTRgene

Construction of Lac and Tet Tether Modified Cas9.

The lactose and tetracycline repressor proteins bind well-definedoperator sequences with high specificity and affinity. GeneTethers arelactose or tetracycline repressor proteins fused with Cas9 (and otherCas proteins), TALENS, or ZNF with that will bind to DNA moleculescontaining the respective operator sequences. Binding ofGeneTether-modified Cas proteins, TALENs, or ZNF to their genomic targetwill thereby physically localize any DNA molecules, bound to therepressor protein fusion, to the same genomic site (FIG. 1).Localization of DNA molecules, homologous to the genomic DNA targetsequences, enhance the efficiency of gene editing by the homologousdirected recombination DNA repair pathways and minimize DNA mutations.

Construction of Lactose Repressor-Modified Cas9

The lactose repressor gene was amplified by polymerase chain reactionfrom Escherichia coli DH5a using lacI Primer 2f/Primer 2r (Table 2) andthe Q5 high fidelity thermostable polymerase (New England Biolabs)according to vendor instructions. The 1083 base pair product wasgel-purified (Monarch DNA Gel Extraction Kit, New England Biolabs) andused for further modifications. Two BbsI restriction endonuclease sitesin the Lactose repressor gene sequence were inactivated using polymerasechain reaction amplification with the mutagenic primers lacIBbs2f,lacIBbs2r, lacIBbs3f, and laciBbs3r to generate A to G transitions incodons 164 and 277, retaining glutamic acid codons (FigureLacIBbsAssembled). Three gel-purified, Q5 high fidelity polymerase,polymerase chain reaction products using primer pairs lacI primer1f/lacI Bbs Primer 2r, lacI Bbs Primer 2f/lacI Bbs Primer 3r, and lacIBbs Primer 3f/lacI Primer 1 r (Table 2) were used to reconstruct thefull length 1083 base pair BbsI-inactivated Lactose repressor proteingene (NEBuilder HiFi Assembly, New England Biolabs).

TABLE 2 PCR Primer DNA Sequences. SEQ ID Primer Sequence NO CF1BfCCTTCTCTGTGAACCTCTATCA 10 CF1f GCAGAGTACCTGAAACAGGA 11 CF5rCATTCACAGTAGCTTACCCA 12 CF7Cr ATAGGAAACACCAAAGATGA 13 CF8CrATAGGAAACACCAATGATAT 14 CF8Crfull ATAGGAAACACCAATGATATTTTCTTTAAT 15GGTGCCAGGC CF9Cf GAAAATATCATTGGTGTTTCCTATGATGAA 16 TATAGATACAG CF96250fTGAGTTAGATGTTTGACGC 17 CF96310f GCTGTGCATTTTCCTCTGGGTAATACTTTA 18 GCF98236f GTCTCTATTACTTAATCTGTACCT 19 CF98328fCTGTGAAGATTAAATAAATTAATATAGTTA 20 AAGCAC CF99287rATGCTCATTCCATTAGGCTATAGTATTA 21 CF99310r CTAATTCTCTGCTGGCAGATCAATGC 22CF101310r CAAGACGTTGTGTTAGGTACATTACATGTA 23 CATC lacI BbsCTCCCATGAGGACGGTACGCGACTGGGC 24 Primer 2f lacI BbsGCCCAGTCGCGTACCGTCCTCATGGGAG 25 Primer 2r lacI BbsGTGGGATACGACGATACCGAGGACAGCTCA 26 Primer 3f TGTTATATC lacI BbsGATATAACATGAGCTGTCCTCGGTATCGTC 27 Primer 3r GTATCCCAC lacI PrimerATGAAACCAGTAACGTTATACGATGTCGC 28 1f lacI Primer TCACTGCCCGCTTTCCAG 29 1rlacI Primer GGTATCCACGGAGTCCCAGCAGCCATGAAA 30 2f CCAGTAACGTTATPrimer1fAge CTGGAGCACCTGCCTGAAATCAC 31 Primer1fXbaCGCGTGCGCCAATTCTGCAGACAAATG 32 Primer1r TGCTGGGACTCCGTGGATACCGACCTTCCG33 CTTC Primer2f GGTATCCACGGAGTCCCAGCAGCCGTGAAA 34 CCAGTAACGTTATPrimer2r GGCGGACTCTGAGGTCCCGGGAGTCTCGCT 35 GCCGCTCTGCCCGCTTTCCAGPrimer3f CGGGACCTCAGAGTCCGCCACACCCGAAAG 36 TGACAAGAAGTACAGCATC Primer3rCGTCCACCTTGGCCATCTCGTTGCTG 37 lacOsymCF1f GTTCGGAATATAAATTGTGAGCGCTCACAA38 TTAAGCTTGCAGAGTACCTGAAACAGGA lacOsymCF5rGTTCGGAATATAAATTGTGAGCGCTCACAA 39 TTAAGCTTCATTCACAGTAGCTTACCCAlacOsymCF5kf GTTCGGAATATAAATTGTGAGCGCTCACAA 40TTAAGCTTGCTGTGCATTTTCCTCTGGGT lacOsymCF5krGTTCGGAATATAAATTGTGAGCGCTCACAA 41 TTAAGCTTCAAGACGTTGTGTTAGGTACATTACATGTAC pUC19polyCF5kr GAATTCGAGCTCGGTACCCGGGGATCCCAA 42GACGTTGTGTTAGGTACATTACATGTAC pUC19polyCF5rGAATTCGAGCTCGGTACCCGGGGATCCCAT 43 TCACAGTAGCTTACCCA tetoCF5kfCACTCCCTATCAGTGATAGAGAAAAGAAAG 44 CTGTGCATTTTCCTCTGGGT tetoCF5krCACTCCCTATCAGTGATAGAGAAACAAGAC 45 GTTGTGTTAGGTACATTACATGTACATC tetoCF1fCACTCCCTATCAGTGATAGAGAAAAGGCAG 46 AGTACCTGAAACAGGA tetoCF5rCACTCCCTATCAGTGATAGAGAAAAGCATT 47 CACAGTAGCTTACCCA 10635fCGGAGCCTATGGAAAAACGCCAGC 48

Construction of the lactose repressor-Cas9 fusion was performed asoutlined in FIG. 8. Three overlapping DNA fragments amplified bypolymerase chain reaction with the Q5 high fidelity polymerase werecloned into AgeI/BgIII digested pSpCas9 BB-2A-GFP(px458) (Genescript,Inc) to generate the GeneTether lactose repressor-Cas9 fusion plasmidpGT1 (FIG. 8D, SEQ ID NO 63) using the NEBuilder system (New EnglandBiolabs). The three, polymerase chain reaction products used for thisassembly were generated using the polymerase chain reaction primers tothe px458 vector Primer1fAge/Primer1r, and Primer3f/Primer3r, and theBbsI-inactivated Lactose repressor gene was amplified usingPrimer2f/Primer2r (Table 2). The resulting plasmid, pGT1, was screenedfor by colony polymerase chain reaction and the Lactose repressor-Cas9fusion gene sequence was confirmed by DNA sequencing (QuintaraBiosciences), restriction endonuclease mapping, and diagnosticpolymerase chain reaction amplification.

Construction of Tetracycline Repressor-Modified Cas9.

The gene encoding the class B TN10 tetracycline repressor, with humancodon preference, was ordered as a synthetic DNA molecule (Genescript,Inc) cloned into the pUC57 vector (FIG. 9 and SEQ ID NO: 65).Construction of the tetracycline-repressor-Cas9 fusion was performedsimilar to the pGT1 lactose repressor-Cas9 fusion (FIG. 10). The three,polymerase chain reaction products used for this assembly were generatedusing the polymerase chain reaction primers to the px458 vectorPrimer1fAge/Primer1r, and Primer3f/Primer3r, and the Tetracyclinerepressor gene was amplified using the primers NLS Linker primer1f/tetlinker Cas9 Primer 1r. The resulting plasmid, pGT9 (SEQ ID NO: 63), wasscreened for by colony polymerase chain reaction and the Tetracyclinerepressor-Cas9 fusion gene sequence was confirmed by DNA sequencing(Quintara Biosciences), restriction endonuclease mapping, and diagnosticpolymerase chain reaction amplification.

Guide RNA Cloning Guide RNA sequences were designed to bind the exon 11gene sequence of the wild type CFTR gene and the CFTR del-F508 mutantgene sequence (Table 3). The guide DNA oligonucleotides were annealed tocreate duplex molecules with Bbs1 compatible overhang and weresubsequently ligated into gel-purified BbsI restriction endonucleasedigested px458, pGT1, and pGT9.

TABLE 3 Guide Sequences. Shown are the DNAoligonucleotides for cloning into the BbsI sitesin pGT1, pGT9, and pX458 Cas9 vectors SEQ Name Sequence ID NOCFWT 130117 fw CACCGATTAAAGAAAATATCATCTT 49 CFWT 130117 fcAAACAAGATGATATTTTCTTTAATC 50 CFWT 130121 rw CACCGAAAGATGATATTTTCTTTAA 51CFWT 130121 rc AAACTTAAAGAAAATATCATCTTTC 52 delF 130127 fwCACCGACCATTAAAGAAAATATCAT 53 delF 130127 fc AAACATGATATTTTCTTTAATGGTC 54delF 130138 rw CACCGACCAATGATATTTTCTTTAA 55 delF 130138 rcAAACTTAAAGAAAATATCATTGGTC 56

aCFTR Donor DNA Generation 5 kilobasepair CFTR DNA fragmentsapproximately centered on the CFTR gene exon 11 were generated bypolymerase chain reaction using the Q5 high fidelity DNA polymerase andprimers CF96310f and CF101310r. Donor DNA fragments containing the CFTRdeIF508 mutation were generated using genomic DNA from the CFBE410-cellline that is homozygous for the deIF508 mutation. Donor DNA fragmentscontaining the wild type (nonmutant) CFTR gene sequence were generatedusing genomic DNA from the HEK293 cell line.

Donor DNA fragments used for gene editing were modified at the 5′ or 3′end of the fragment to contain either the lactose operator sequence(AATTGTGAGCGCTCACAATT, SEQ ID NO: 57) or tetracycline operator sequence(CACTCCCTATCAGTGATAGAGAAA SEQ ID NO: 58). To generate 500 base pairdonor DNA molecules, polymerase chain reaction amplification using theQ5 high fidelity thermostable DNA polymerase with the primer pairslacOsymCF1f/CF5r to add the lactose operator sequence to the 5′ end ofthe donor DNA or primer pairs Cf1f/lacOsymCF5r to add the lactoseoperator to the 3′ end of the donor DNA fragment (Figure Donor DNAMolecules). Donor DNA molecules with the tetracycline operator sequencewere generated using the primer pairs tetoCF1f/CF5r and CF1f/tetoCF5r.Donor DNA fragments were gel-purified prior to use in cell transfectionsfor gene editing.

Cell Transfection with Px458, pGT1, pGT9 Cas9 Vectors and 500 Base PairDonor DNA Fragments

HEK293 cells were transfected in 12-well plates using Lipofectamine 3000(Invitrogen, Inc) with 500 ng of plasmid DNA and 500 ng of gel purifieddonor DNA fragments. The px458, pGT1, and pGT9 plasmids contain a greenfluorescent protein reporter gene that is expressed in transfected cellsand allows monitoring of transfection efficiencies. Green fluorescentprotein expression was visualized by fluorescence microscopy atapproximately 48 hours post transfection. At 48-72 hours posttransfection, 12-well plates of transfected cells were washed once withphosphate buffered saline and stored at −80° C. until DNA was harvestedfor analysis of gene editing efficiencies.

Analysis for the deIF508 Mutation by Allele-Specific Polymerase ChainReaction Analysis

The presence of the deIF508 mutation in a population of CFTR wild typecells can be detected using polymerase chain reaction with Taqpolymerase and the primer pair CF1 Bf/CF8Cr. Since the CF1 Bf primer islocated 5′ or outside of the donor DNA fragment, the 388 basepairproduct is generated from an “inside-out” approach and is specificallydiagnostic for gene edited events and will not amplify randomlyintegrated or unintegrated donor DNA molecules.

Genomic DNA was prepared directly from each well of a 12-well plate(Genejet Genomic Purification Kit, ThermoFisher, Inc) and DNAconcentration determined by ultraviolet spectroscopy. Allele specificpolymerase chain reactions were performed on 100 ng of genomic DNA and500 μM primer, 1.5 mM MgCl₂, HotStart Taq polymerase (New EnglandBiolabs). The thermocycler program for semiquantitative amplification ofthe deIF508 mutant DNA sequence was 95° C., 2 minutes; 35 cycles of 95°C., 30 seconds, 50° C., 30 seconds, 72° C., 1 minute; followed by 72°C., 8 minutes. The 388 base pair polymerase chain reaction product wasvisualized with ultraviolet light on a 1.5% agarose gel stained withGelred (Biotium, Inc). For semi-quantification of the PCR product,standard genomic DNA samples with varying ratios of wild type anddeIF508 mutant DNA were amplified in parallel to experimental DNAsamples.

Cell Transfection

Two different tethers (lactose-repressor-Cas9 fusion or tetracyclinerepressor-Cas9 fusion) were used in combination with donor DNA moleculescontaining a 5′ lactose or tetracycline operator sequence or a 3′lactose or tetracycline operator sequence (FIGS. 11 and 12) andcontaining the deIF508 deletion sequence. Homology directed repair byhomologous recombination between the genomic CFTR target Cas9-induceddouble strand break and donor fragment would catalyze transfer of thedeIF508 DNA sequence to the genomic target (FIG. 12).

Cas9 guides complementary to the nonmutated wild type CFTR exon 11 DNAsequence, but not to the deIF508 DNA sequence were selected to allowrecognition of the genomic DNA target and prevent the Cas9 enzyme fromrecognizing, and cleaving, the deIf508 donor DNA fragment.

Approximately 5×10⁵ HEK293 cells per well were seeded, 24 hours beforetransfection, into Corning 12-well tissue culture plates such that thewells were 50-80% confluent at the time of transfection. The media onthe cells was changed prior to transfection.

For transfections, 500 ng of px458, pGT1 or pGT9 was mixed with 500 ngof donor DNA fragment in 50 μL of DMEM medium. 2 μL of the P3000 reagentwas then added to the DNA/DMEM solution, mixed well, and allowed toincubate at room temperature for 5 minutes. A solution of Lipofectamine3000 was made by adding 1.5 μL of undiluted Lipofectamine 3000 to 50 μLof DMEM and equal volumes of DNA/DMEM solutions and Lipofectamine/DMEMsolutions were mixed and incubated at room temperature for 12-15minutes. 100 μL of the DNA/P3000/Lipofectamine solution was then addedper well of Corning 12-well plates. Two days after transfection thecells are examined with fluorescent microscopy to assess the extent ofsuccessful transfection.

Gene Tethers Increase Gene Editing Efficiency

Several variables for the effect of GeneTether modified Cas9 vectors ongene editing efficiency were tested: a lactose repressor-Cas9 fusionprotein, tetracycline repressor-Cas9 fusion protein, Cas9 guidescomplementary to the top or bottom strand of genomic target, and 500 bpdonor DNA molecules containing the deIF508 deletion with the lactoseoperator sequence at the 5′ or 3′ end of the donor DNA fragment.

The combination of pGT1 vector (lactose repressor-Cas9 fusion) with the130117 guide (genomic target forward strand) and lactose operator on the3′ end of donor DNA (FIG. 13, lane 7) demonstrates higher gene editingefficiency as compared to the px458 vector with unmodified Cas9 and samedonor DNA fragment (compare 388 basepair products lane 3 and lane 7,FIG. 13. Measurement of band intensities using ImageJ software(https://imagej.nih.gov) indicates that the tethered donor DNA complexhas an approximate 7-fold higher gene editing efficiency compared to thepx458 unmodified Cas9. Indeed, comparison of lane 7 band intensity tothe reconstructed mixture of WT:deIF508 DNA (lanes 14-19) suggests thedeIF508 mutation is present in approximately 10% of the genomic DNA.Since transfection efficiencies for these experiments were 10%, or less,up to 100% of cells transfected with the combination ofpGT1/130117guide/3′ lactose operator sequence underwent successful geneediting.

Other combinations of pGT1/guide/donor DNA performed equal to or,slightly better than, unmodified Cas9 (FIG. 13; lane 9 vs lane 5) forgene editing, demonstrating that the Cas9 protein activity was notaffected by the lactose repressor fusion. The placement of the lactoseoperator sequence may slightly favor the 3′ placement (FIG. 13; lanes 7and 9 vs lanes 3 and 5). Donor DNA transfected with px458 not containingguide sequence showed low levels of gene editing (FIG. 13; lanes 10 and11). Some faint 388 bp product is evident in lanes 10, 12, and 13 thatis likely due to artifactual low-level mispriming of PCR primers usedfor the allele-specific PCR.

Gene editing with the pGT9 tetracycline repressor-Cas9 fusion vector,guide sequences, and donor fragments did not yield appreciably bettergene editing frequencies than the control px458 vector (FIG. 14, lanes2-5 vs lanes 6-9). The placement of the tetracycline at the 3′ end ofthe donor DNA fragment appears to result in slightly better gene editingefficiencies (FIG. 14 lanes 2, 4, 6, 8 vs lanes 3, 5, 7) similar toplacement of the lactose operator sequence.

These results demonstrate that the GeneTether lactose repressor-Cas9fusion protein encoded by pGT1 can significantly increases gene editingefficiency as compared to the unmodified Cas9 found in the px458 vector.Indeed, it is possible that the combination of pGT1/130117 guide/deIF508lacO 500 successfully caused gene editing of WT to deIF508 in almost100% of transfected cells.

1. A composition for tethering donor DNA to a nuclease, the compositioncomprising a nucleic acid comprising donor DNA and a consensus sequencefor a DNA binding domain; and at least one of: a fusion proteincomprising a nuclease coupled to a DNA binding domain for binding theconsensus sequence; and a nucleic acid encoding the fusion protein. 2.The composition of claim 1 wherein the wherein the nuclease is a Casprotein, a Transcription activator-like effector nuclease (TALEN), ameganuclease, a Zinc Finger or a MADzyme.
 3. The composition of claim 1wherein the nuclease is a Cas protein.
 4. The composition of claim 3wherein the Cas protein is Cas9
 5. The composition of claim 1 whereinthe fusion protein further comprises a nuclear localization sequence. 6.The composition of claim 2, further comprising a guide RNA thatinteracts with the Cas protein and a target DNA sequence.
 7. Thecomposition of claim 1 wherein the consensus sequence comprises the Lacoperator (SEQ ID NO: 66), the TRP operator (SEQ ID NO: 68), the TEToperator (SEQ ID NO 67), the GAL-4 binding site (SEQ ID NO: 1), or theIHF binding site (SEQ ID NO 2).
 8. The composition of claim 7 whereinthe consensus sequence comprises a sequence with at least 80%, 85%, 90%,95% or at least 99% identity to the Lac operator, the TRP operator, theTET operator, the GAL-4 binding site, or the IHF binding site
 9. Thecomposition of claim 1 wherein the DNA biding domain comprises the LACrepressor, TET repressor, TRP-repressor, GAL-4, or IHF, or a portionthereof sufficient to bind the consensus sequence.
 10. The compositionof claim 1 wherein the DNA binding domain is the LAC repressor,preferably amino acids 43-403 of SEQ ID NO
 9. 11. The composition ofclaim 1 wherein the nuclease is coupled to the DNA binding domain via alinker.
 12. The composition of claim 11 wherein the linker comprises asequence selected from any one of SEQ ID Nos: 3 to 7, a GGS linker, oramino acids 404-419 of SEQ ID NO
 9. 13. The composition of claim 1wherein the fusion protein comprises the LAC repressor and Cas9.
 14. Thecomposition of claim 1 comprising a vector, wherein the vector compriseseither or both of: a. the nucleic acid comprising the donor DNA and theconsensus sequence for a DNA binding domain; and b. the nucleic acidencoding the fusion protein.
 15. The composition of claim 14 wherein thevector further comprises a nucleic acid sequence encoding a guide RNAthat interacts with the Cas protein and a target DNA sequence.
 16. Thecomposition of claim 1 wherein the fusion protein is nuclease deficient.17. An isolated host cell comprising the composition of claim
 1. 18. Amethod for editing DNA in a cell, the method comprising; contacting thecell with the composition of claim 1 under conditions suitable for theinteraction of the fusion protein with a target DNA sequence.
 19. Amethod for editing DNA in a cell, the method comprising a) contactingthe cell with the composition of claim 16 under conditions suitable forthe interaction of the fusion protein with a first target DNA sequence;and b) contacting the cell with a nucleic acid editing system adapted toedit the genomic DNA at a second target DNA sequence, under conditionssuitable for nucleic acid editing.
 20. The method of claim 18 whereinthe target DNA sequence is selected from genomic DNA, mitochondrial DNA,viral DNA, or exogenous DNA.
 21. The method of claim 18 wherein theefficiency of editing is at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90% at least 95%, or at least 99%.
 22. The method of claim 18wherein the cell is in a subject, preferably a human subject.
 23. A kitcomprising: a nucleic acid comprising donor DNA and a consensus sequencefor a DNA binding domain; and at least one of a fusion proteincomprising a nuclease coupled to a DNA binding domain for binding theconsensus sequence; and a nucleic acid encoding the fusion protein. 24.The kit of claim 23 wherein the nuclease is a Cas protein, aTranscription activator-like effector nuclease (TALEN), a meganuclease,a Zinc Finger or a MADzyme.
 25. The kit of claim 24 wherein the Casprotein is Cas9.